You are hereSeptember 23, 2011 | Pluripotent Stem Cells
An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells
Using a mouse hybridoma library raised against undifferentiated hESCs and a library of commercially available monoclonal antibodies (mAbs), binding was compared between undifferentiated hESCs and 3 day differentiated hESCs (via retinoic acid or BMP4 treatment) in order to identify any antibodies which may separate these two populations. One mAb, designated SSEA-5, effectively labelled undifferentiated hESCs with this labelling diminishing greatly during differentiation; indeed to an even greater extent than the previously established pluripotency-associated cell surface markers TRA-1-81, SSEA-3 and SSEA-4. SSEA-5+ cells were also shown to have a greater expression of POU5F1, NANOG and SOX2 than SSEA-5-, and in addition the SSEA5 mAb was able to label the pluripotent cells of the inner cell mass of in vitro fertilization-derived blastocyst-stage human embryos. SSEA-5 was then tested against a range of differentiated cell types to examine the exclusivity of labelling. In hESC-derived teratomas, SSEA-5 was expressed in only a subset of epithelial structures expressing SSEA-4 and epithelial-specific antigen (ESA), amounting to approximately 2% of the teratoma cells, which exhibited morphologies reminiscent of primordial hPSCs, suggesting teratoma stem cells. Therefore, these SSEA-5+ cells were extracted and were shown through injection under the kidney capsule of immunodeficient mice to form large teratomas in all assays undertaken (7 of 7), while SSEA-5- transplants gave rise to very small teratomas in 3 of 11 assays. In addition, SSEA-5 labelling was not evident upon analysis of a panel of 12 human tissues from 7-month-old fetuses, indicating the specificity of the anti-SSEA-5 mAb to pluripotent stem cells and suggesting its capability to remove residual teratoma-initiating cells.
Attempts to indentify the SSEA-5 antigen indicated that SSEA-5 was not a single protein antigen and the researchers went on to identify SSEA-5 as a glycan moiety, as anti-SSEA-5 was found to specifically bind to glycans with a terminal presentation known as the H type-1 (H-1) antigen. Importantly, SSEA-5 was found not to bind any glycan without the H-1 motif and binding did not overlap with commercial mAbs against H-2, SSEA-3 and SSEA-4. Further analysis of glycan expression in differentiating pluripotent stem cells (hESCs and hiPSCs) indicated that there was a shift from H-1 like glycans to H-2 type glycans, further suggesting that H-1 glycans to which SSEA-5 binds are enriched in pluripotent populations and the change to H-2 type glycans in differentiated hESCs provides further data demonstrating the usefulness of the anti-SSEA-5 mAb in removing pluripotent cell types from differentiating cultures.
Functional assays were then implemented to assess the effectiveness of using SSEA-5 to remove teratoma-forming cells from differentiating cultures. Undifferentiated hESCs were spiked at a 1:100 ratio into fully differentiated cells and live cells sorted from this mixture caused large teratomas in eight replicates within 7 weeks. However, when the population was depleted of SSEA-5+ cells only small tumours in 3 of 8 replicates were observed. Next, the ability of anti-SSEA-5 to remove teratoma forming cells from partially differentiated cultures was tested by comparing the teratoma-forming potential of high SSEA-5 vs. low SSEA-5 expressing cells from 3 day retinoic acid treated hESCs. Teratomas formed from both populations, but those derived from SSEA-5-high cells were significantly larger compared to those formed from SSEA-5-low cells. Both these functional assays demonstrate that SSEA-5 can enrich for pluripotent cells, however, they also show that the anti-SSEA-5 mAb is insufficient by itself to separate all residual pluripotent and potentially teratoma forming cells. Further experiments were therefore carried out to analyse whether other antibodies could synergise with SSEA-5 to achieve complete removal of pluripotent cells. Initial analyses uncovered 5 antibodies (CD9, CD30, CD50, CD90 and CD200) which demonstrated similar expression dynamics to SSEA-5 during ESC differentiation with CD9, CD30 and CD90 previously shown to correlate with pluripotency. Multicolour flow cytometry analysis demonstrated that a single population co-expressing high levels of CD9, CD50, CD90 and SSEA-5 decreased in proportion during differentiation to 52% to 6% at days 3 and 10 of retinoic acid mediated differentiation. Further expression analyses of CD9, CD30, CD90 and CD200 (CD50 probes were insensitive and SSEA-5 is a glycan) revealed that >99% of the non-pluripotent tissues tested did not express these 4 markers, whereas almost all pluripotent samples did, with their specificity declining with the sequential removal of any of these markers. Immunohistochemistry of 7-month-old human fetal tissues revealed that approximately half of the organs analyzed were positively labeled with three or more of these markers, but labeled structures within tissues rarely overlapped, overall suggesting that concurrent high expression of three of these markers is rarely found in non-pluripotent tissues. SSEA-5/CD9/CD90-high and SSEA-5/CD50/CD200-high populations purified from differentiating hESC cultures formed large teratomas with evidence of three germ layers, while the corresponding negative fraction did not. Notably, all growths emerging from the SSEA-5/CD9/CD90-low population exhibited histological evidence of only epithelial and mesenchymal derivatives, and lacked evidence of structures typical of teratomas, including bone, cartilage and neural rosettes. In comparison, both TRA-1-81/SSEA-4-high and TRA-1-81/SSEA-4-low populations formed teratomas consisting of tissues of all three germ layers, suggesting that these two pluripotency-associated antibodies are not able to purify teratoma-forming cells.
Overall this exciting piece of research demonstrates the ability to remove teratoma-formation potential from incompletely differentiated hESC cultures, through the separation of residual teratoma-forming cells. This has obvious beneficial impact for the clinical transplantation of hESC-derived cells and tissues. However, the presence of small outgrowths from the SSEA-5/CD9/CD90-low and SSEA-5/CD50/CD200-low fractions of cells does suggest that additional markers need to be sought to reduce this risk further and the inability of TRA-1-81 and SSEA-4 to purify teratoma-forming cells suggests that more novel antibodies, such as have been generated in this study, may be required. Further, the discovery of the anti-SSEA-5 mAb also gives us a new tool with which to label pluripotent cells, expressed in higher levels than most pluripotency cell surface markers utilised to date.
Ben-David U, Benvenisty N.
The tumorigenicity of human embryonic and induced pluripotent stem cells.
Nat Rev Cancer. 2011 Apr;11(4):268-77.
Cao F, Drukker M, Lin S, Sheikh AY, Xie X, Li Z, Connolly AJ, Weissman IL, Wu JC.
Molecular imaging of embryonic stem cell misbehavior and suicide gene ablation.
Cloning Stem Cells. 2007 Spring;9(1):107-17.
Choo AB, Tan HL, Ang SN, Fong WJ, Chin A, Lo J, Zheng L, Hentze H, Philp RJ, Oh SK, Yap M.
Selection against undifferentiated human embryonic stem cells by a cytotoxic antibody recognizing podocalyxin-like protein-1.
Stem Cells. 2008 Jun;26(6):1454-63.
Schuldiner M, Itskovitz-Eldor J, Benvenisty N.
Selective ablation of human embryonic stem cells expressing a "suicide" gene.
Stem Cells. 2003;21(3):257-65
Tan HL, Fong WJ, Lee EH, Yap M, Choo A.
mAb 84, a cytotoxic antibody that kills undifferentiated human embryonic stem cells via oncosis.
Stem Cells. 2009 Aug;27(8):1792-801.
Tang C, Drukker M.
Potential barriers to therapeutics utilizing pluripotent cell derivatives: intrinsic immunogenicity of in vitro maintained and matured populations.
Semin Immunopathol. 2011 Apr 11.
Tang C, Lee AS, Volkmer JP, Sahoo D, Nag D, Mosley AR, Inlay MA, Ardehali R, Chavez SL, Pera RR, Behr B, Wu JC, Weissman IL, Drukker M.
An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells.
Nat Biotechnol. 2011 Aug 14.